1. Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC)
Good afternoon everyone.
My talk today is on the work I carried out as part of the development of an EGFR and KRAS testing service for NSCLC at the Aberdeen lab.
Joel Tracey1, Caroline Clark1, Christine Bell1, Keith Kerr2, Marianne Nicholson3, Aileen Osborne1, Zosia Miedzybrodzka1, Kevin Kelly1
1Department of Medical Genetics, Polwarth Building, Aberdeen Royal Infirmary, Aberdeen
2Department of Pathology, Aberdeen Royal Infirmary, Aberdeen
3Clinical Oncology, Aberdeen Royal Infirmary, Aberdeen

2. Non Small Cell Lung Cancer (NSCLC)
Lung cancer is one of the most commonly diagnosed types of cancer in the UK
Leading cause of cancer-related death in both men and women
Non-Small Cell Lung Cancer ~ 80% (3)
Adenocarcinoma (ADC)
Squamous cell carcinoma (SCC)
Large cell carcinoma (LCC)
Percentage of NSCLC subtypes in UK
In the UK, Lung cancer is the leading cause of cancer related death in both men and women
Of the cases diagnosed 80% are NSCLC
NSCLC can be divided into 3 histological subtypes: ADC, SCC, LCC

3. EGFR and KRAS in NSCLC
Acquired mutations in the EGFR and KRAS genes are important in the development of NSCLC
Mutations result in inappropriately activated proteins – tumour cells become ‘addicted’ to growth signals
EGFR and KRAS mutations most common in adenocarcinoma
EGFR and KRAS mutations are mutually exclusive
EGFR and KRAS are two tyrosine kinases that are important in the development of NSCLC
Both are involved in cell signaling pathways that control cell proliferation and survival
Acquired mutations in the EGFR and KRAS genes can result in constitutively activated proteins which can lead to uncontrolled cancer cell growth
These mutations most commonly occur in adenocarcinomas and are mutually exclusive

4. Treatment of NSCLC Surgery - possible in about 20% of cases (4)
Cytotoxic chemotherapy and/or radiotherapy - mostly ineffective
Survival rate poor (7% alive 5 years after diagnosis) (4)
Tyrosine Kinase Inhibitors (TKI) – new type of chemotherapeutic agent – fewer side-effects than cytotoxic chemotherapy
Target and block growth factor signals – e.g. Epidermal Growth Factor Receptor (EGFR)
Current cancer treatments such as surgery, cytotoxic chemotherapy and radiotherapy are largely ineffective in treating NSCLC and as such the survival rate is low
TKIs are a new type of treatment which could offer a significant improvement over conventional chemotherapeutics
Many TKIs act by targeting and blocking growth factor signals such as those mediated by EGFR

5. EGFR Tyrosine Kinase Inhibitors
Erlotinib (Tarceva) and Gefitinib (Irresa)
EGFR targeted TKIs can be used for treatment of NSCLC patients with somatic activating EGFR mutations
Mutations within EGFR TK domain enable TKIs to bind with greater affinity
Patients with activating EGFR mutations have a better response to TKI therapy and improved survival
TKI
A number of EGFR TKIs have been developed
Clinical trials have shown them to be effective specifically in treating patients with somatic activating mutations in the EGFR gene
Mutation positive patients have a better initial response to TKI therapy and improved survival
Conversely KRAS mutations are associated with a lack of response to TKI therapy
Patients with KRAS mutations show little or no response to TKI treatment

6. Clinical Trial – IPASS Study
EGFR mutation +ve (M+) patients respond better to TKI therapy than chemotherapy but....
EGFR mutation –ve patients (M-) have a poorer response to TKI therapy than chemotherapy!!!
Probability of Progression Free Survival
Time (months)
The results of the recent IPASS clinical trial illustrate well the importance of knowing a patients EGFR mutation status
This study compared the response of chemo-naïve EGFR mutation +ve and –ve patients to the EGFR TKI Gefitinib and the chemotherapeutic Carboplatin.
The graph shows that EGFR +ve patients (solid lines) responded better to Gefitinib than Carboplatin
However, the EGFR –ve patients (dashed lines) had a much poorer response to Gefitinib than Carboplatin
This shows that is it important to identify all patients with EGFR mutations however it is equally important to avoid false positive results!

7. EGFR mutations
Mutations in the EGFR gene are present in 10-15% of NSCLC patients
And as the diagram on the left shows the mutations mainly occur between Ex 18 to 21
The majority of mutations are associated with sensitivity to TKIs - the two most common mutation types being exon 19 deletions and the L858R mutation in exon 21.
Some EGFR mutations, mainly those in exon 20, are associated with resistance to TKIs
KRAS mutations occur in about 30% of NSCLC patients and are mainly found in codons 12, 13 and 61
Mutations in the EGFR gene found in 10-15% of NSCLC patients (5, 6)
Exon 19 deletions & L858R (Exon 21) make up 85-90% of all mutation +ve cases (7)
Exon 20 mutations (e.g. T790M) commonly resistance mutations

8. KRAS mutations KRAS mutations occur in ~30% of NSCLC tumours (8)12 13 61 C A G T
Codons
Wt seq
Multi-variable mutations
KRAS mutations occur in ~30% of NSCLC tumours (8)
Codon 12 most common mutation site

9. Project Aims Develop and set-up methods for EGFR and KRAS analysis
Determine best methods for analysis of EGFR and KRAS mutations
Develop and validate appropriate methodologies for testing
The aims of this project were to:
Initially develop and set-up methods for EGFR and KRAS analysis
Determine the best methods for analysis of EGFR and KRAS mutations
And further develop and validate the appropriate methods for testing

10. Samples 48 Adenocarcinoma patient samples (ARI Pathology Dept)
4 EGFR +ve control samples (Holland)
All were FFPE lung tumour samples (cores, slides & rolls)
14 DNA samples for KRAS analysis (Transgenomic Inc)
Mutations in codon 12, 13 and 61
Varied mutation levels (3% to 33%)
During this study we received 48 patient samples from the ARI Path dept as well as 4 EGFR +ve controls
All of which were formalin fixed paraffin-embedded lung tumour samples
We also received 14 DNA samples specifically for KRAS analysis from Transgenomic

11. Challenges with NSCLC testing using FFPE samples
Frequently low sample quantity = Low DNA yield
Variable tumour content within sample (<5% to 100%)
Poor DNA quality - Degradation (<300bp), PCR inhibitors
Genetic heterogeneity – inter- and intra-tumour variation
Pathology departments involvement at this stage important to maximise % tumour – macro-dissection
We found there were a number of challenges with NSCLC testing using FFPE samples:
Low sample quantity, variable tumour content, poor DNA quality and tumour genetic heterogeneity can all impact on the ability to detect somatic mutations

12. Methodology Plan
Extract DNA from FFPE lung tumour samples (Dewax, phenol/chloroform)
Quantify all DNA samples (Nanodrop)
PCR amplification using specific primers (in-house/published)
Quantify all DNA samples
PCR amplification using specific primers
EGFR
Direct Sequencing
WAVE HS dHPLC + fragment collection
WAVE Surveyor
Ex19 Fragment Length Analysis
Ex21 Pyrosequencing
KRAS
Direct Sequencing
WAVE Surveyor
Pyrosequencing
EGFR exons and KRAS codons 12, 13 and 61 PCR products were subject to various methods of analysis
In addition to direct sequencing, high sensitivity WAVE dHPLC and WAVE Surveyor methods, pyrosequencing and fragment length analysis

13. Principles of Methods Used
WAVE HS dHPLC – Partially denaturing, High sensitivity by fluorescent detection (x100), mutation identified by presence of mutant/WT heteroduplex peaks
Fragment collection – After passing through detector eluted DNA fragments were collected in vials at 30s intervals
WAVE Surveyor – Enzymatic method, detects DNA mismatches, WAVE size separation, mutation identified by presence of cleavage products
In the WAVE Surveyor method mismatches in mutant/WT heteroduplexes are cleaved by a restriction enzyme and by using size separation on the WAVE system mutations are identified by the presence of smaller cleavage products
The high sensitivity WAVE dHPLC method uses fluorescent labeling and detection of DNA to give greater sensitivity than the standard UV detection. In this partially denaturing method mutants are detected by the presence of heteroduplexes

14. Principles of Methods Used
Fragment Length Analysis – FAM labelled PCR products, size separation on ABI 3130, analysis using Gene Marker software
Pyrosequencing – Real-time sequence data, Pyrophosphate (PPi) substrate for reaction cascade, light produced measured – relative to nucleotides incorporated
Fragment length analysis was performed by using FAM-labeled primers for PCR. The labeled PCR products were then size separated on the ABI 3130
The pyrosequencing method is a real-time semi-quantitative sequencing method which utilises the pyrophosphate generated during nucleotide incorporation as a substrate for an enzymatic reaction. Light is produced which is relative to the number of nucleotides incorporated.

15. Summary of KRAS Results
Blind study (Transgenomic samples)
Pyrosequencer detected all mutations in Transgenomic samples (lowest = 3%)
2 samples not detected by the WAVE Surveyor method (3%)
6 samples were below the detection limit of sequencing (<10%)
33% (16/48) of patient samples positive for KRAS mutations – tested by both pyrosequencing and direct sequencing
The results from the KRAS part of this study confirmed that the pyrosequencer is currently the best method available for KRAS analysis…..something which I think many labs have already found
It was more sensitive and quicker than both the WAVE surveyor method and direct sequencing
33% of the patients tested were positive for a KRAS mutation which is consistent with published figures
Percentage of KRAS mutations identified (by codon)

16. EGFR Results Sample: EGFR +ve Control
Mutation: Exon 19 Del (c del; p.L747 – T751del)
+ve control
Sequencing WT
267
298
257
Size control
+ve control WT
174
Uncleaved product
WAVE Surveyor
102 80
This is an example of the EGFR results we obtained with the different methods trialed
All three results show the same exon 19 deletion
The mutation is indicated by the presence of additional low level sequence in the sequencing result,
The heteroduplex peak in the WAVE dHPLC result,
And the presence of cleavage products in the WAVE Surveyor result
+ve control WT
WAVE HS dHPLC

17. Enrichment of EGFR mutant by WAVE dHPLC + fragment collection
Ex 19 WT
Ex 19 del (direct seq)
The following results we feel show that its possible to enrich for mutant fragments using fragment collection in conjunction with the WAVE dHPLC system
The top chromatogram is the wild-type reference sequence.
The middle chromatogram is the direct sequencing result for an exon 19 deletion mutant, the mutant sequence is visible as low level peaks
The bottom chromatogram is for the same sample sequenced after fragment collection and repeat PCR. We can see the level of the deletion mutant sequence has been increase enough that the mutation surveyor software has identified and indicated the mutation
Ex 19 del (enriched by fragment collection + repeat PCR)
Sequences analysed with Mutation Surveyor software (Soft Genetics)

18. Additional methods Ex 19 Fragment length analysis
Ex 19 del
Ex 19 WT
Ex 19 Fragment length analysis
G863D
L861Q
L858R
The other methods tested looked specifically at one exon each.
Fragment analysis was used to detect exon 19 deletions only. The smaller deletion fragment is indicated by the red arrow in this case.
The pyrosequencing method developed was used to detect exon 21 mutations. In this result we can see an L858R mutation at a level of about 28% in the sample
Ex 21 Pyro-sequencing
L858R mutant

19. Summary of EGFR Results
12.5% (6/48) of patient samples positive for EGFR mutation
(Ex Deletions; Ex Insertion; Ex L858R)
All methods detected Ex19 del mutations in 4 EGFR +ve control samples
WAVE Surveyor confirmed all mutations found by direct sequence analysis
One Ex19 del mutant too low to report by direct sequence analysis but clear by WAVE Surveyor and Fragment length analysis
Pyrosequencer – successfully detected Ex21 mutants
Confident no false positive results
All EGFR +ve samples were KRAS –ve
Mutations confirmed by multiple methods
To summarise the EGFR results:
We found 6 out of 48 patients tested had an EGFR mutation
All methods detected the mutations in the positive control samples
The WAVE surveyor method successfully detected all mutations found by direct sequencing
In addition one mutation missed by direct sequence analysis was detected by WAVE surveyor and fragment analysis methods which would indicate these methods to be more sensitive than direct sequencing
The pyrosequencing method successfully detected all exon 21 mutants previously found
Overall we feel confident that no false positive results would have been reported as:
All EGFR pos samples were confirmed as KRAS neg and the results were confirmed by multiple methods

20. Comparison of EGFR methods
WAVE HS dHPLC
SURVEYOR
Sequencing
Fragment analysis (Ex19 only)
Pyrosequencing (Ex 21 only)
Hands on time *
2hr 30min
2hr 45min
1hr 30min
2hr 15min
Cost (per sample)
£16.50
£15.60
£26.70
£0.57
£8.04
Results analysis time*
45min
30min
Total Time to result *
40hr 40min
24hr 45min
11hr 15min
6hr
4hr 35min
Sample required
120ng
20ng
Detection Limit ?
~4-5%
~10%
3-5%
A comparison of the performance and costs of each method was also made. The key findings were:
There is little difference in hands on times and sample requirements for the various methods
However compared to sequencing, the WAVE methods cost less per sample and results analysis time is less
Fragment analysis is an inexpensive and quick method of screening EGFR exon 19
Pyrosequencing is more expensive per sample but it is a quick and sensitive method for EGFR exon 21 analysis
Times based on analysis of 15 samples
Cost per sample does not include staff costs

21. Conclusions
Pick-up rate of EGFR mutations consistent with published data
Direct sequencing pick-up rate higher than expected. This likely to be due to enrichment of samples for tumour tissue by macro-dissection
WAVE Surveyor, fragment analysis and pyrosequencing methods may be useful as a higher sensitivity screen in conjunction with direct sequencing
Fragment collection is a viable method for enrichment of low level mutations
To conclude:
The overall pick-up rate of EGFR mutations was consistent with published data
The direct sequencing pick-up rate was higher than expected as only one mutation was missed but this could likely be due to enrichment of the samples by macro-dissection prior to genetic analysis
The WAVE surveyor, fragment analysis and pyrosequencing methods are all promising and may be useful as higher sensitivity screening methods in conjunction with direct sequencing
Our results also suggest fragment collection could be a viable method for enrichment of low level mutants

22. Current Testing Strategy
NSCLC Patient (M/F, smoker/non-smoker)
SCC / LCC
Adenocarcinoma
Not Tested
Assessment of tumour content and macrodissection
Pathology
Molecular Genetics
EGFR Ex PCR
KRAS codons 12, 13 and 61 PCR
Finally, this chart shows the current EGFR/KRAS testing strategy at the Aberdeen lab.
Adenocarcinomas are selected and macro-dissected in pathology and then tested for EGFR and KRAS mutations in parallel by various methods for each gene.
Parallel testing by multiple methods is being employed to minimise turn-around times but also for greater confidence in the results
Direct Sequencing /Pyrosequencing
Direct Sequencing/WAVE Surveyor/Fragment Length Analysis
Report

23. Acknowledgements Aberdeen Lab Clinical/Pathology Caroline Clark
Christine Bell
Aileen Osborne
Louise Carnegie
Heather Greig
Kevin Kelly
Transgenomic
Gerald Martin
Clinical/Pathology
Keith Kerr
Marianne Nicholson
Zosia Miedzybrodzka
Astra Zeneca
For providing funding

24. References
1 Ferlay J. et al. Estimates of the cancer incidence and mortality in Europe in Annals of Oncology (2007) 18:
2 Harkness E.F. et al. Changing trends in incidence of lung cancer by histologic type in Scotland. Int. J. Cancer (2002) 102:
3 D’Addario G. & Felip E. Non-small-cell lung cancer: ESMO Clinical Recommendations for diagnosis, treatment and follow-up. Annals of Oncology (2008) 19 (Sup 2): ii39 - ii40
4 Scottish Executive Health Department. Cancer scenarios: an aid to planning cancer services in Scotland in the next decade The Scottish Executive: Edinburgh.
5 Janne P.A. et al. A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res (2006); 12 (3):
6 Sequist L.V. et al. Epidermal Growth Factor Receptor mutation testing in the care of lung cancer patients. Clin Cancer Res (2006); 12 (Sup 14): 4403s – 4408s
7 Sequist L.V. & Lynch T.J. EGFR Tyrosine Kinase Inhibitors in lung cancer: an evolving story. Ann Rev Med (2008) 59:
8 Do, H. et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS detection in formalin fixed paraffin embedded biopsies. BMC Cancer (2008) 8:142